Phosphoglycosyl transferases (PGTs) are membrane-bound enzymes, which catalyze the transfer of a phosphoglycosyl group from an activated UDP-sugar donor to a polyprenylphosphate acceptor. PGT-catalyzed reactions afford amphiphilic polyprenyldiphosphate-linked carbohydrates, thereby committing glycan assembly processes to a location at the membrane interface. PGTs are specific for the identity of the UDP-sugar that they act on, which defines the nature of the transferred phosphoglycosyl group. These enzymes are most commonly involved in an essential early step that features in many glycoconjugate assembly pathways including those that lead to bacterial lipopolysaccharides and capsular polysaccharides, teichoic acids, and N- and O-linked glycoproteins. Such glycoconjugates play critical roles in the interactions of microbial pathogens with the hosts that they infect, however, there is a dearth of selective small molecule inhibitors, which can be used as tools to assess and define the significance of specific glycoconjugates in microbial physiology and to elucidate their roles in host-pathogen interactions. This research will address the critical need for new chemical agents as inhibitors for bacterial PGTs and seeks to provide selective inhibitors of diverse PGTs that act on different UDP-sugar donor substrates. In this exploratory program we will employ synergistic approaches involving inhibitor design and synthesis, X-ray based structure determination, and enzymatic analysis to develop a general method for the genesis of potent and selective bacterial PGT inhibitors. The central theme of the research approach relies on exploiting the molecular logic of the natural product nucleoside antibiotics, in particular tunicamycin, as a blue print for the structure-guided design and synthesis of a new family of modular chemical structures that act as selective inhibitors of PGTs. Tunicamycin is a potent bisubstrate analog inhibitor of the bacterial PGT, Wec A, which is a homolog of the yeast PGT Alg7, and also inhibits PglC-Hpu, a small PGT from H. pullorum with IC50 of 35030 nM. These enzymes catalyze phosphoglycosyl transfer from UDP-GlcNAc to undecaprenylphosphate thereby initiating lipopolysaccharide and N-linked glycoprotein biosynthesis in the respective organisms. Focusing first on three small PGTs from different microbial pathogens that act on diverse phosphoglycosyl donor substrates (UDP-GlcNAc, UDP-Gal and UDP-Bac) and a common undecaprenyl phosphate (UndP) acceptor, we will develop and validate tractable and modular synthetic platforms for the assembly of new inhibitors. If successful, this research will lead to a new inhibitor design approach that can be applied broadly to a large family of PGTs and to powerful chemical tools that will afford valuable new insight into the functional significance of complex glycoprotein conjugates that decorate the cell surfaces of many microbial pathogens.